Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor

AbstractThe complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human γ-trace, human salivary acidic protein and chicken egg-white cystatin

The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis

AbstractThe superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21 500. The sequence of B. gingivalis SOD showed 44–51% homology with those for iron-specific SODs (Fe-SODs) and 40–45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65–74%) or Mn-SOD family (42–60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families

Overexpression and Divalent Metal Binding Properties of the Methionyl Aminopeptidase from \u3cem\u3ePyrococcus furiosus\u3c/em\u3e

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP\u27s is Fe(II). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest Km values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (kcat/Km) of PfMetAP-II for MP-p-NA at 30 °C was 799 500 and 340 930 M-1 s-1 for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (Kd) for the first metal binding site were found to be 50 ± 15 and 20 ± 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 ± 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be ∼85 °C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 °C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and Km values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25−85 °C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 °C are as follows:  ΔG⧧ = 46.23 kJ/mol, ΔH⧧ = 10.79 kJ/mol, and ΔS⧧ = −119.72 J·mol-1·K-1 for Co(II)-loaded PfMetAP; ΔG⧧ = 46.44 kJ/mol, ΔH⧧ = 16.94 kJ/mol, and ΔS⧧ = −99.67 J·mol-1·K-1 for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (\u3e50 °C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 °C) than Co(II)-loaded PfMetAP-II